Browsing by Subject "Mycobacterium tuberculosis"
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Item Characterization of Mycobacterium tuberculosis Cor, a Protein Essential for Carbon Monoxide Resistance and Pathogenesis(2015-01-28) Zacharia, Vineetha Mariam; Hendrixson, David R.; Alto, Neal; McDonald, Jeffrey; Shiloh, MichaelTuberculosis, caused by Mycobacterium tuberculosis, is global health burden as it remains one of the most devastating human infectious diseases causing two million deaths annually and latently infecting a third of the world's population. We previously demonstrated that M. tuberculosis induces an enzyme, heme oxygenase (HO1), that produces carbon monoxide (CO) gas and that M. tuberculosis adapts its transcriptome during CO exposure. We now demonstrate that M. tuberculosis carries a novel resistance gene to combat CO toxicity. We screened an M. tuberculosis transposon library for CO-susceptible mutants and found that disruption of Rv1829 (carbon monoxide resistance, cor) leads to marked CO sensitivity. Heterologous expression of Cor and Cor homologue from Thermotoga maritima (TM0160) in Escherichia coli rescued it from CO toxicity, suggesting a conserved function across diverse microbial species. Importantly, the virulence of the cor mutant is attenuated in a mouse model of tuberculosis. Thus, Cor is necessary and sufficient to protect bacteria from host-derived CO. Evolutionary modeling suggested that Cor forms an active site, thus we predicted that Cor has enzymatic activity. To determine potential Cor enzymatic activity, we profiled the mycobacterial metabolome using liquid-chromatography mass spectrometry and, in vitro, monitored metabolite fluctuations in the presence and absence of recombinant Cor. Our activity-based metabolomic profiling data showed the accumulation of phosphatidic acid and multiple phospholipids in the presence of Cor, indicating its potential role in catalyzing a reaction involved in phospholipid biosynthesis. Our in vivo metabolomic analysis of a Cor mutant strain showed that in the presence of CO, levels of dihydroxyacetone phosphate is increased, whereas levels of glycerol-3-phosphate is reduced, which is required for phospholipid biosynthesis. Furthermore, we identified key metabolic enzymes in Mtb that physically interact with Cor using bioinformatics and immunoprecipitation techniques. Specifically, Cor interacted with glycerol-3-phosphate dehydrogenase 2, an enzyme that catalyzes the interconversion of glycerol-3-phosphate to dihydroxyacetone phosphate. Taken together, this represents the first report of a role for HO1-derived CO in controlling infection of an intracellular pathogen and the first identification of a CO resistance gene in a pathogenic organism, which may have a critical role in phospholipid biosynthesis.Item Development of a Lipidomics Platform to Discover Enzymatic Activities of Mycobacterial Proteins of Unknown Function(2020-05-01T05:00:00.000Z) Pasko, Breanna Lauren; Alto, Neal; Shiloh, Michael; Sperandio, Vanessa; McDonald, JeffreyMycobacterium tuberculosis (Mtb), a facultative intracellular pathogen, is responsible for 10 million new cases of tuberculosis and 1.5 million deaths annually. Mtb infection can be acute but also lifelong. Indeed, despite the devastating global impact on health, how Mtb is able to adapt to the human body and survive for years is unknown. To better understand the physiology of Mtb infection, exploring the functions of individual proteins produced by Mtb is critical. Less than half of the proteins encoded in the M. tuberculosis genome have been fully characterized either directly through experimentation or indirectly through annotation. Many of these unknown genes likely encode proteins important for pathogenesis and adaptation to host responses. I created a novel method to facilitate the identification of novel substrates and products using direct infusion tandem mass spectrometry (DI-MS/MS) analysis of nonpolar metabolite fluctuations after transient and rapid protein overexpression. I studied 24 proteins using this lipidomic method. Of these, several demonstrate unique patterns of metabolites secondary to rapid induction of protein expression, and work towards identifying their specific enzymatic activities is ongoing. I selected one protein, Cor (Rv1829), for further characterization as it was recently shown to be fundamental to carbon monoxide (CO) resistance and pathogenesis of Mtb during host infection. To study Cor in vitro, I purified recombinant Cor from E. coli and used activity based metabolic profiling (ABMP) to measure changes in mycobacterial metabolites exposed to Cor. In the ABMP assay, exposure of mycobacterial metabolites to Cor led to consumption of acetyl phosphate and accumulation of phosphatidic acid (PA). I developed direct enzymatic assays for Cor activity, and confirmed the consumption of acetyl phosphate. To assess substrate binding, I used isothermal titration calorimetry (ITC) and demonstrated binding of Cor to acetyl phosphate. Additionally, Cor interacted directly with cardiolipin (CL), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), and sulfatide on membrane lipid strips. I overexpressed Cor in M. smegmatis and Mtb and lipids were extracted and analyzed through DI-MS/MS to assess changes in lipid composition. I combined in vivo lipidomics and in vitro ABMP to biochemically characterize Cor and found that Cor is interacting with bacterial lipids. In vivo lipidomic analysis revealed an accumulation of PA and PG by 3 hours. Our findings suggest that Mtb Cor is interacting with lipids involved in the phospholipid biosynthesis pathway and modifying bacterial lipid composition by accumulating PG. These experiments provide mechanistic insight into the enzymatic function of Cor. Furthermore, the metabolomics and lipidomics approaches developed in this study can be broadly applied to study proteins of unknown function from other organisms.Item Don't cough on me!: airborne transmission of disease(2018-03-23) Shiloh, Michael U.Item Identification of Scavenger Receptor B1 as the Airway Microfold Receptor for Mycobacterium tuberculosis(2020-12-01T06:00:00.000Z) Khan, Haaris Suwaid; Hooper, Lora V.; Schmid, Sandra; Kohler, Jennifer J.; Farrar, J. DavidMycobacterium tuberculosis (Mtb) can enter the body through multiple routes, including via specialized transcytotic cells called microfold cells (M cell). However, the mechanistic basis for M cell entry remains undefined. Here, I show that M cell transcytosis depdends on the Mtb Type VII secretion machine and its major virulence factor EsxA. I identify scavenger receptor B1 (SR-B1) as an EsxA receptor on airway M cells. SR-B1 is required for Mtb binding to and translocation across M cells in mouse and human tissue. Together, my data demonstrate a previously undescribed role for Mtb EsxA in mucosal invasion and identify SR-B1 as the airway M cell receptor for Mtb.Item Identification of Smaller Noncoding RNAs Produced by Mycobacterium Tuberculosis in Infected Macrophages That Regulate Mtb Growth and Survival(2021-12-10) Coskun, Fatma Sevde; Hancks, Dustin C.; Conrad, Nicholas; Winter, Sebastian E.; van Oers, Nicolai S. C.It is estimated that one-third of the world's population is infected with Mycobacterium tuberculosis (Mtb). While much work has focused on the role of different proteins encoded by Mtb in pathogenesis, recent studies have revealed that Mtb also transcribes many noncoding RNAs whose functions remain poorly characterized. A subset includes small RNAs (sRNAs) between the sizes of 50-350 nts. The current study focused on the identification and characterization of miRNA-like sRNAs <50 nts produced by Mtb. A sRNA-centered RNA-sequencing approach was performed and a subset of Mtb-encoded smaller noncoding RNAs (sncRNAs) were identified. Thirty-five distinct Mtb-encoded sncRNAs were discovered, with most being induced in infected eukaryotic cells. Three sncRNAs, sncRNA-1, sncRNA-6, and sncRNA-8, predominated the read counts. They were contained in longer RNA transcripts with stable secondary RNA stem loops and structures like precursor microRNAs. My work established that sncRNA-1 positively regulates two mycobacterial transcripts involved in oleic acid biosynthesis. Loss- and gain- of-function approaches reveal that sncRNA-1 enhances Mtb growth and survival in nutrient-depleted cultures as well as in infected macrophages. Given evidence that RNA processing enzymes were involved in the formation of the sncRNAs, different components of core RNA degradosome were characterized for their ability to process the precursor forms of the sncRNAs. My work revealed that PNPase degrades sncRNA-8 and preliminary evidence suggests that sncRNA-1 is also likely a target, which could be critical in the oleic acid deficient media. Overall, my study reveals that Mtb produces a set of sncRNAs in infected cells, with one modulating mycobacterial gene expression and mycobacterial pathogenicity coupled to oleic acid biogenesis. Future studies will address the functions of other sncRNAs and focus on the identification of sncRNA processing enzymes.Item Mechanisms of Macrophage Detection and Control of Mycobacterium tuberculosis Infection(2016-04-15) Collins, Angela Christine; Wakeland, Edward K.; Shiloh, Michael; Hansen, Eric J.; Marciano, Denise; Russell, David W.; Yarovinsky, FelixMacrophages use different mechanisms to recognize and respond to Mycobacterium tuberculosis infection. Macrophage recognition of M. tuberculosis is characterized by the production of a robust type I interferon response dependent on the activation of a cytosolic surveillance pathway by the recognition of M. tuberculosis DNA in the cytosol. The DNA sensor recognizing M. tuberculosis DNA and initiating activation of the cytosolic surveillance pathway has yet to be defined. Here we describe a role for the recently characterized DNA sensor cGAS in the detection of M. tuberculosis infection and initiation of the type I interferon response as well as a role for cGAS in the targeting of M. tuberculosis to the autophagosome. We demonstrate that cGAS deficiency is associated with decreased survival in a mouse model of M. tuberculosis infection. The second part of this thesis explores how macrophages respond to M. tuberculosis infection. We previously showed that in mice M. tuberculosis infection induces the expression of the carbon monoxide producing enzyme heme oxygenase (HO1) in the macrophage and that the CO is sensed by M. tuberculosis to initiate a dormancy program. Mice deficient in HO1 succumb to M. tuberculosis infection more readily than wild-type (WT) mice. While the mechanisms used by mouse macrophages to control intracellular M. tuberculosis infection, including nitric oxide synthase, the respiratory burst, acidification and HO1 are well studied, how human macrophages control M. tuberculosis infection is less well understood. Here we show that HO1 is induced by and colocalizes with M. tuberculosis in both mouse and human tuberculosis lesions, and that M. tuberculosis induces and colocalizes with HO1 during human macrophage infection in vitro. HO1 enzymatic activity in human macrophages is necessary for inflammatory cytokine production and for control of intracellular M. tuberculosis replication. Finally, we find that a polymorphism in the HO1 promoter is associated with susceptibility to human tuberculosis. Thus, we demonstrate an important role for HO1 in controlling human tuberculosis.Item Mycobacterium Tuberculosis Virulence Factor Mpt64 Targets the Endoplasmic Reticulum(2019-04-10) Stamm, Chelsea Elizabeth; Sperandio, Vanessa; Shiloh, Michael; Alto, Neal; Winter, Sebastian E.Mycobacterium tuberculosis, the causative agent of tuberculosis, is one of the most successful human pathogens. One reason for its success is that M. tuberculosis can reside within host macrophages, a cell type that normally functions to phagocytose and destroy infectious bacteria. However, M. tuberculosis is able to evade macrophage defenses in order to survive for prolonged periods of time. Many intracellular pathogens secret virulence factors targeting host membranes and organelles to remodel their intracellular environmental niche. I hypothesized that M. tuberculosis secreted proteins that target host membranes are vital for M. tuberculosis to adapt to and manipulate the host environment for survival. Thus, I characterized nearly 200 secreted proteins from M. tuberculosis for their ability to associate with eukaryotic membranes using a live-dead, temperature sensitive yeast screen and to manipulate host trafficking pathways using a modified inducible secretion screen. I identified five M. tuberculosis secreted proteins that both associated with eukaryotic membranes and altered the host secretory pathway. One of these secreted proteins, Mpt64, localized to the endoplasmic reticulum during M. tuberculosis infection of murine and human macrophages and impaired the unfolded protein response in macrophages. These data highlight the importance of secreted proteins in M. tuberculosis pathogenesis and provide a basis for further investigation into their molecular mechanisms.Item The Pivotal Role of cGAS in Host Defense Against Mycobacterium Tuberculosis and in Particle-Induced Immunity(2017-11-02) Cai, Haocheng; Fu, Yang-Xin; Hooper, Lora V.; Yan, Nan; Chen, Zhijian J.The cGAS-cGAMP-STING pathway is a major DNA sensing pathway in the cytosol. The DNA sensing mechanism is crucial for host defense against bacteria, viruses, and parasites. In the previous study from our lab, the cGAS-STING pathway has been identified as being critical for antiviral activities. However, anti-bacterial activity is more complicated due to the complexity of the invading agents. Mycobacterium tuberculosis (Mtb) is a pandemic pathogen that triggers millions of deaths every year. It is discovered that Mtb ejects its DNA component into host cytosol. We have uncovered the cGAS pathway, which elicits type I interferons and autophagy in macrophages and other cell types, as a defending mechanism against Mtb infection. cGAS is critical for mouse survival against chronic Mtb infection. In conclusion, cGAS is critical for the host defense against Mtb, especially in the induction of type I interferon and autophagy. Previous studies revealed that some fusogenic liposomes and enveloped virus-like particles can activate type I interferons. Here we have shown that fusogenic liposomes and nonenveloped virus-like particles can activate the cGAS pathway and trigger interferon production, which is associated with the amount of cytosolic DNA. Moreover, cGAS and STING are critical for the humoral immune response elicited by virus-like particles. Overall, we have delineated the significant role of cGAS in mediating the immune response induced by fusogenic liposomes and virus-like particles. Additionally, we have proven that cGAS heterozygous mice have largely retained the antiviral activity in herpes simplex virus infection, compared with wildtype mice. This has shed light on the development of cGAS inhibition therapies against autoimmune diseases. Overall, this dissertation has identified the pivotal roles of the cGAS-STING DNA sensing pathway in Mycobacterium tuberculosis infection and particle-induced immunity, as well as the effect of cGAS heterozygosity on antiviral activities.Item [Southwestern News](2004-03-22) O'Brien, StephenItem Using Gene Overexpression as a Potential Method to Elucidate Antibiotic Resistance Mechanisms in Mycobacteria(2015-03-31) Bacalao, Maria Alexandra; Gumbo, Tawanda; Van Oers, Nicolai S. C.; Kant, Shashi; Wakeland, Edward K.Mycobacterium Tuberculosis remains a major public health threat, with 9 million new cases and 1.5 million deaths in 2013. Also concerning is the rise of antbiotic resistance, leading to the development of Multiple Drug Resistant (MDR) and Extensively Drug Resistance (XDR) Mycobacterium tuberculosis strains. The development of these strains is multifactorial involving both extrinsic factors and intrinsic factors. Extrinsic factors include treatment noncompliance and a delay in diagnosis with existing drug sensitivity methods. Intrinsic factors include heterogeneity in the bacterial population and localization in cavitations and other areas where antibiotic penetration is difficult. Our study analyzed the potential role of a potential mechanism of antibiotic resistance, overexpression of existing wild type M. tuberculosis genes involved in mycobacterial antibiotic defense. We cloned seven M. tuberculosis wild type genes using PCR and the subcloned those constructs into E. Coli. Further subcloning steps placed these genes into shuttle vectors that allowed for transformation into M tuberculosis and M. smegmatis. The M. Smegmatis mutant overexpressing the GyrA gene was selected for dose-response studies to establish whether GyrA overexpression led to increased fluoroquinolone resistance. Two M. smegmatis GyrA mutant and one wild type strain were incubated with increasing concentration of Moxifloxacin. Initial dose-response studies did not show yield an adequate dose-response curves under the study conditions. However, the MIC was higher for the GyrA mutant strains than for wild type M. smegmatis, showing that these GyrA mutants were likely more resistant than wild type and that the role of gene overexpression in M. tuberculosis antibiotic resistance merits further study.